An interesting aspect of natural cytotoxicity by NK cells is that several pairwise combinations of receptors, which have very distinct signaling properties, result in potent synergy for activation of intracellular Ca2+ release, cytokine production, and cytotoxicity. The main question we have addressed was how distinct signals from co-activation receptors are integrated to regulate such synergy, and which molecular checkpoint(s) control this process. Among the receptor combinations that provide synergistic activation in resting NK cells are NKG2D (CD314) and 2B4 (CD244). Upon stimulation with its natural ligands, such as ULBP molecules, NKG2D recruits phosphatidylinositol-3-kinase (PI3K) and Grb2-Vav1 through its association with DAP10. 2B4 binds to its ligand CD48, which is expressed on hematopoietic cells, and recruits the small adaptor SAP bound to the tyrosine kinase Fyn through cytoplasmic tyrosine-based motifs. Because synergy requires the integration of such diverse signals, understanding the basis for synergy would disclose how disparate signals converge to a certain point at which synergy occurs. Synergistic NK cell activation by two coactivation receptors was accompanied by enhanced Vav1 phosphorylation, which was equivalent to the sum of phosphorylation induced by each receptor alone and was required to overcome inhibition by c-Cbl ubiquitin ligase. At present, there is no information about how distinct signals from synergizing receptors converge to regulate Vav1 and its downstream signaling. Based on the additive, rather than synergistic phosphorylation of Vav1 during synergy, we hypothesized that different pools of Vav1 may complement each other to achieve synergy. As a first step, we tested the role of adapters known to contribute to signaling for lymphocyte activation. We investigated whether adaptor proteins such as SLP-76 and linker for activation of T cells (LAT) are required for NK cell synergy. SLP-76 and LAT are important adaptors that form the backbone of signaling complexes T cells. Synergy among receptors is best achieved by complementation of independent signals. Stimulation of natural killer (NK)-cell cytotoxicity and cytokine secretion requires synergistic signals from co-activation receptors. Our previous work has shown that synergy is required to overcome inhibition of guanine exchange factor Vav1-dependent signals by the ubiquitin ligase c-Cbl. Whether complementation of unique signals is involved and, if so, at what level, is unknown. We have shown here that the adapter SLP-76 is required for the synergy and that each receptor of a synergistic pair controls the phosphorylation of a specific tyrosine in SLP-76. Each one of the two phosphorylated tyrosines that constitute separate binding sites for Vav1 is required for synergistic mobilization of Ca2+. The selective phosphorylation of either tyrosine 113 or tyrosine 128 in SLP-76 is unique to signaling by natural cytotoxicity co-activation receptors, as stimulation of NK cells by the Fc receptor CD16 resulted in phosphorylation at both sites, much like T cells stimulated by T-cell receptor. Our data reveal an unexpected degree of selectivity in the phosphorylation of two SLP-76 tyrosines by NK cell co-activation receptors, suggest that binding of two Vav1 molecules to SLP-76 may have a synergistic effect on Vav1 function, and show that triggering of NK cell effector function is tightly regulated by complementary signals. In studies of activation of lymphocytes most of the attention has been given to the signaling receptors on lymphocytes, and how these receptors are distributed at the plasma membrane once they bind to their ligands on antigen-presenting cells (APC) or target cells. In contrast, whether the distribution of ligands on the stimulating cells has an impact on lymphocyte responses has received little attention. Furthermore, while the importance of membrane microdomains in receptor-mediated activation of lymphocytes has been established, much less is known about the role of receptor ligand distribution within membrane domains on APC and target cells. Detergent-resistant membrane (DRM) domains, into which glycophosphatidylinositol (GPI)-linked proteins partition, are enriched in cholesterol and glycosphingolipids. ULBP1 is a GPI-linked ligand for natural cytotoxicity receptor NKG2D. NKG2D is one the main co-activation receptors on NK cells. To investigate how ULBP1 distribution on target cells affects NKG2D-dependent NK cell activation independently of HLA class I ligands for inhibitory receptors, we expressed ULBP1 in a mouse cell line. A chimera consisting of the extracellular portion of ULBP1 and the transmembrane region of CD45 was generated. Its expression resulted in the localization of the normally GPI-linked ULBP1 from detergent-resistant membrane fractions to detergent-soluble fractions. Clustering and lateral diffusion of ULBP1 was not affected by changes in the membrane anchor. This redistribution of ULBP1 caused a significant reduction in cytotoxicity and degranulation by NK cells, implying a role for receptor ligand distribution in the activation of NK cell responses. Our data suggests that ligand distribution into distinct membrane domains in general may play an underappreciated role in the activation of NK cells. Given the potential of tumor cells or virus-infected cells to alter ligand distribution at the plasma membrane and to escape immune responses, it will be important to investigate how the distribution of other ligands impacts the activation of lymphocytes. Although vesicle exocytosis has been extensively studied in endocrine and neuronal cells, much less is known about the fusion of lytic granules in cytotoxic lymphocytes. Imaging and electrophysiological techniques have suggested that both complete and incomplete fusion occur in endocrine cells and several different neural synapses. To date, the precise steps of lytic granules fusion in cytotoxic lymphocytes such as CTL and NK cells have not been imaged in live cells. To test whether complete fusion or incomplete fusion of lytic granules with the plasma membrane occurs in cytotoxic lymphocytes, we used the human NK cell line NKL, as well as primary NK cells from human peripheral blood. We used TIRF microscopy to examine lytic granules labeled with fluorescently tagged Fas ligand in the NK cell line NKL stimulated with phorbol ester and ionomycin and in primary NK cells activated by physiological receptorligand interactions. FasL is present in the lytic granule membrane of CTL and NK cells. pHluorin is a pH sensitive variant of the green fluorescence protein (GFP) and has been used to monitor vesicle fusion in endocrine and neuronal cells. We attached pHluorin to the C-terminal, lumenal portion of Fas ligand (FasL), and DsRed to the N-terminal, cytosolic tail of FasL. By specifically labeling lytic granules with different fluorescent proteins, and by using TIRF microscopy, we have shown that NK cells use two distinct modes for exocytosis of lytic granules: complete fusion, characterized by loss of granule content and rapid diffusion of Fas ligand at the plasma membrane;and incomplete fusion, characterized by transient fusion pore opening and retention of Fas ligand at the fusion site. Thus, we conclude that lytic granules in NK cells undergo both complete and incomplete fusion with the plasma membrane, and propose that incomplete fusion may promote efficient recycling of lytic granule membrane after the release of cytotoxic effector molecules.